The formation of pituitary hormones from precursor molecules and degradation of peptide hormones and factors regulating pituitary function is dependent upon the action of specific proteolytic enzymes. The long term objective of this project is to identify, isolate and characterize these enzymes and to determine their role in the process of formation and degradation of peptide hormones and biologically active peptides. Our work in the last two years has led to the discovery in the pituitary of a multicatalytical protease complex of high molecular weight. The complex behaves in solution as a single protein, but possesses trypsinlike, chymotrypsinlike and peptidylglutamyl-peptide bond hydrolyzing activities, each of which is associated with a separate component of the complex. The complex generates enkephalin and enkephalin - containing peptides from precursor molecules and may be involved in the formation of several hormones. We wish to study the biochemistry and function of this complex. Attempts will be made to dissociate the complex, to isolate its components, and to determine their primary and secondary specificity. Knowledge acquired from the specificity studies will be used as a basis for the synthesis of specific, active site directed inhibitors, that should provide a tool for the examination of the role of the complex in pituitary function. We have also identified and partially characterized three other pituitary proteases: a membrane-bound metalloendopeptidase that cleaves bonds on the amino side of hydrophobic amino acids and favors the presence of a pair of basic amino acids on the N-terminal side of the attacked bond; a prolylendopeptidase that hydrolysis bonds on the carboxyl side of proline residues and degrades biologically active peptides including luteinizing hormone-releasing hormone and thyrotropin-releasing hormone; and a soluble thiol-protease with a high affinity toward substrates with basic amino acid residues. The properties, specificity and function of these enzymes will be further studied, and inhibitors will be synthesized, that should be used to test the role of the enzymes in pituitary function. The action of the enzymes on pituitary hormones will be determined. Experiments will be performed to detect any changes of proteolytic activity related to altered secretory function of the pituitary, induced by accepted procedures.